Reagent for immunoassay, and device using the same

ABSTRACT

A reagent for use in an immunoassay for measuring haptens, antigens or antibodies by means of a competitive binding method, which comprises a combination of an antibody and a labelled hapten or a labelled antigen or a combination of a hapten or an antigen and a labelled antibody, wherein the antibody and the labelled hapten or the labelled antigen in one combination or the hapten or the antigen and the labelled antibody in another combination are capable of undergoing reversible binding, and a device for use in an immunoassay wherein the reagent of the present invention is included in a single container. 
     An immunoassay can be performed in a short time by the use of the immunoassay device of the present invention.

This application is a divisional of application Ser. No. 07/480,165,filed on Feb. 14, 1990, now U.S. Pat. No. 5,283,176, the entire contentsof which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

This invention relates to a reagent for use in an immunoassay and animmunoassay device in which the reagent is included in a container, formeasuring haptens, antigens or antibodies by means of a competitivebinding method.

Backed by the recent rapid progress in immunology and geneticengineering, various methods, as well as assay kits, for the measurementof trace substances in the body have been developed making use of immunereactions.

Such immune reaction-aided measurements are divided chiefly into asandwich method and a competitive binding method on the basis of themeasuring principles.

A principle of the sandwich method, in the case of an antigen as asubstance to be detected for example, is to capture the antigen byholding (sandwiching) it between an antibody (insoluble antibody) whichhas a specific affinity for one of the antigenicity-active sites(determinant group) of the antigen molecule and another antibody(labelled antibody) which also has a specific affinity but for anotherdeterminant group of the antigen molecule and then to measure thequantity of the antigen in the sample based on the signals originatingfrom the labelled antibody. In consequence, the test substance has to bean antigen which has at least two specific determinant groups.

Many studies have been performed on the sandwich method, which isgenerally used for the measurement of high molecular weight substances,such as proteins, polypeptides, saccharides, lipids and their complexes.For example, since the reagent for use in the measurement of antigenscomprises an insoluble antibody and another labelled antibody, and theseinsoluble and labelled antibodies do not react with each other ifpresent together, an assay kit in which both of these antibodies arepreviously included in a single container has been developed (JapanesePatent Public Disclosure No. 57-136165). In addition, making use of thisassay kit, an easily operable automatic measuring device in which animmune reaction can be started by simply adding a test sample into acontainer has been developed.

In the convenient competitive binding method, in the case of an antigenas a substance to be detected for example, the antigen in a sample and alabelled antigen are competitively bound to an antibody, and thequantity of the antigen in the sample is then measured based on thesignals originating from the labelled antigen. In consequence, thesubstance to be tested may be either an antigenic substance having onlyone determinant group or an antibody prepared from such an antigenicsubstance, or a hapten and an antibody which is prepared by using animmunogenic compound composed of a hapten and an immunoactive carrier.

The competitive binding method is generally used for the measurement ofsubstances which are difficult to be analyz by the sandwich technique,for example, substances having relatively low molecular weights, such assteroids, amines, amino acids and peptides.

In the convenient competitive binding method, however, theantigen-antibody reaction is irreversible. Therefore, in the case of anantigen as a substance to be detected for example, accurate measurementcan be achieved when a labelled antigen is added into a container, inwhich an insoluble antibody is previously included, only after, or atthe same time as, the addition of a sample into the container. Inconsequence, it has been generally considered that an insoluble antibodyand a labelled antigen for use in an immune reaction could not beincluded together in one container prior to the addition of a sample.

As described above, the competitive binding method is a useful techniquefor the measurement of trace substances having relatively low molecularweights. However, complex operations are required in the case of manualmeasurement, because an insoluble antibody and a labelled antigen foruse in an immune reaction have to be included in separate containers,and the labelled antigen held in the separate container has to beinjected only after, or at the same time as, the addition of a sampleinto the container in which the insoluble antibody is previouslyincluded. In addition, a system for keeping and injecting a labelledantigen for every measuring item is required in the case of an automaticmeasuring device, which has been an obstacle to the development of anautomatic device for the measurement of multiple items.

For the purpose of solving such a problem, a method for including bothan insoluble antibody and a labelled antigen in a single containermaking use of a freeze-drying technique has been proposed (JapanesePatent Public Disclosure No. 62-148857 and Japanese Patent PublicDisclosure No. 62-151758). However, these prior patents do not disclosestabilities of the insoluble antibody and labelled antigen with thepassage of time when the method is applied to practical operation.Therefore, a problem of stabilities with the passage of time stillremains unsolved.

SUMMARY OF THE INVENTION

In view of the above, it therefore is an object of the present inventionto provide an immunoassay reagent making use of the principle of thecompetitive binding method, which comprises a combination of areversibly bindable insoluble antibody and a labelled hapten, acombination of a reversibly bindable insoluble antibody and a labelledantigen, a combination of a reversibly bindable insoluble hapten and alabelled antibody, or a combination of a reversibly bindable insolubleantigen and a labelled antibody.

Another object of the present invention is to provide a device for usein an immunoassay wherein any one of the above-described immunoassayreagents is included in a single container.

In one form of the present invention, there is provided a reagent foruse in an immunoassay for measuring a hapten, comprising

(a) a labelled hapten comprising a first hapten and a labelling agentbound to said first hapten, and

(b) an antibody prepared by using an immunogen comprising a secondhapten and an immunoactive carrier bound to said second hapten,

wherein said first and second haptens are individually selected from thegroup consisting of the hapten to be measured and derivatives thereof,and said labelled hapten (a) and said antibody (b) are capable ofundergoing reversible binding; or a reagent for use in an immunoassayfor measuring a hapten, comprising

(c) a first hapten, and

(d) a labelled antibody comprising an antibody (b) prepared by using animmunogen comprising a second hapten and an immunoactive carrier boundto said second hapten, and a labelling agent bound to said antibody,

wherein said first and second haptens are individually selected from thegroup consisting of the hapten to be measured and derivatives thereof,and

said first hapten (c) and said labelled antibody (d) are capable ofundergoing reversible binding.

In a second form of the present invention, there is provided a reagentfor use in an immunoassay for measuring an antigen, comprising

(e) a labelled antigen comprising a first antigen and a labelling agentbound to said first antigen, and

(f) an antibody prepared by using an immunogen of second antigen,

wherein said first and second antigens are individually selected fromthe group consisting of the antigen to be measured and derivativesthereof, and said labelled antigen (e) and said antibody (f) are capableof undergoing reversible binding; or a reagent for use in an immunoassayfor measuring an antigen, comprising

(g) a first antigen, and

(h) a labelled antibody comprising an antibody (f) prepared by using animmunogen of a second antigen, and a labelling agent bound to saidantibody,

wherein said first and second antigens are individually selected fromthe group consisting of the antigen to be measured and derivativesthereof, and

said first antigen (g) and said labelled antibody (h) are capable ofundergoing reversible binding.

In a third form of the present invention, there is provided a reagentfor use in an immunoassay for measuring an antibody, comprising

(a) a labelled hapten comprising a first hapten and a labelling agentbound to said first hapten, and

(b) an antibody prepared by using an immunogen comprising a secondhapten and an immunoactive carrier bound to said second hapten,

wherein said first and second haptens are individually selected from thegroup consisting of the hapten to be measured and derivatives thereof,and said labelled hapten (a) and said antibody (b) are capable ofundergoing reversible binding; or a reagent for use in an immunoassayfor measuring an antibody, comprising

(c) a first hapten, and

(d) a labelled antibody comprising an antibody (b) prepared by using animmunogen comprising a second hapten and an immunoactive carrier boundto said second hapten, and a labelling agent bound to said antibody,

wherein said first and second haptens are individually selected from thegroup consisting of the hapten to be measured and derivatives thereof,and

said first hapten (c) and said labelled antibody (d) are capable ofundergoing reversible binding.

In a fourth form of the present invention, there is provided a reagentfor use in an immunoassay for measuring an antibody, comprising

(e) a labelled antigen comprising a first antigen and a labelling agentbound to said first antigen, and

(f) an antibody prepared by using an immunogen of second antigen,

wherein said first and second antigens are individually selected fromthe group consisting of the antigen to be measured and derivativesthereof, and said labelled antigen (e) and said antibody (f) are capableof undergoing reversible binding; or a reagent for use in an immunoassayfor measuring an antibody, comprising

(g) a first antigen, and

(h) a labelled antibody comprising an antibody (f) prepared by using animmunogen of a second antigen, and a labelling agent bound to saidantibody,

wherein said first and second antigens are individually selected fromthe group consisting of the antigen to be measured and derivativesthereof, and

said first antigen (g) and said labelled antibody (h) are capable ofundergoing reversible binding.

In a fifth form of the present invention, there is provided a device foruse in an immunoassay wherein any one of the above-described immunoassayreagents is included in a single container.

The term "hapten" as used herein refers to any commonly called"incomplete antigen" which is inactive itself as an immunogen regardlessof the presence of a determinant group, but becomes active when it islinked to an immunoactive carrier. Also, the term "antigen" as usedherein refers to any commonly called "complete antigen" which is activeas an immunogen as it is.

Other objects and advantages of the present invention will be madeapparent by the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a, 1b, 1c and 1d are graphs showing results of a study on theexistence of reversible immune reaction between anti-estrogen antibodiesand labelled estrogen derivatives.

FIG. 2 is a graph showing results of a study on the reaction timerequired for the completion of equilibrium state in the immune reactionfor the measurement of estrogen.

FIGS. 3a, 3b, 3c and 3d are graphs showing results of a study on theexistence of reversible immune reaction between anti-estradiolantibodies and labelled estradiol derivatives.

FIG. 4 is a graph showing results of a study on the reaction timerequired for the completion of equilibrium state in the immune reactionfor the measurement of estradiol.

FIGS. 5a, 5b, 5c and 5d are graphs showing results of a study on theexistence of reversible immune reaction between anti-progesteroneantibodies and labelled progesterone derivatives.

FIG. 6 is a graph showing results of a study on the reaction timerequired for the completion of equilibrium state in the immune reactionfor the measurement of progesterone.

DETAILED DESCRIPTION OF THE INVENTION

Any immunoassay of the prior art is performed based on the assumptionthat an immune reaction proceeds in the direction of the formation ofantigen-antibody complexes and the reaction is almost irreversible.

However, the inventors of the present invention have predicted that, ifa combination of an antibody and a hapten is used in which the immunereaction reaches its equilibrium state quickly and binding of theantibody and the hapten is reversible, a new competitive binding methodcould be established making use of the principle of the commonly usedcompetitive binding method. In such a new competitive binding method, acombination of an insoluble antibody and a labelled hapten or acombination of an insoluble hapten and a labelled antibody, for use inan intended immune reaction for the immunoassay of haptens, may beincluded previously in a single container. Such a new competitivebinding method may also be usable even when an immune reaction occursbetween the antibody and the hapten during the preservation period,because, due to the reversible nature of the reaction, the labelledhapten molecules once bound to the insoluble antibody molecules or thelabelled antibody molecules once bound to the insoluble hapten moleculeswill be unbound again in proportion to the amount of hapten molecules ina sample newly injected into the container and, instead, the haptenmolecules in the sample will bind to the antibody molecules.

In the same manner, the inventors of the present invention havepredicted that, if a combination of an antibody and a hapten or anantigen is used in which the immune reaction reaches its equilibriumstate quickly and binding of the antibody and the hapten or the antigenis reversible, a new competitive binding method could be establishedmaking use of the principle of the commonly used competitive bindingmethod. In such a new competitive binding method, a combination of aninsoluble hapten or an insoluble antigen and a labelled antibody oranother combination of an insoluble antibody and a labelled hapten or alabelled antigen, for use in an intended immune reaction for theimmunoassay of antigens or antibodies, may be included previously in asingle container. Such a new competitive binding method may also beusable even when an immune reaction occurs between the insolubleantibody and the labelled hapten or the labelled antigen or between theinsoluble hapten or insoluble antigen and the labelled antibody duringthe preservation period, because, due to the reversible nature of thereaction, the labelled antigen molecules once bound to the insolubleantibody molecules or the labelled antibody molecules once bound to theinsoluble antigen molecules will be unbound again in proportion to theamount of antigen molecules in a sample newly injected into thecontainer and, instead, the antigen molecules in the sample will bind tothe antibody molecules, or the labelled antibody molecules once bound tothe insoluble hapten molecules or the antigen molecules (or the labelledhapten molecules or the labelled antigen molecules once bound to theinsoluble antibody molecules) will be unbound again in proportion to theamount of antibody molecules in a sample newly injected into thecontainer and, instead, the antibody molecules in the sample will bindto the hapten molecules or the antigen molecules.

The present invention has been accomplished by finding such combinationsof antibodies and haptens or antibodies and antigens in which the immunereaction proceeds reversibly and reaches its equilibrium state quickly.

The immunoassay reagent of the present invention is useful for themeasurement of specific substances, such as haptens, antigens andantibodies, in a sample by means of a competitive binding method.

The measuring principle is described below, firstly taking the case of ahapten as an example of the substance to be measured.

When a substance to be measured is a hapten, the reagent of the presentinvention is divided into two cases: "case 1" being a combination of alabelled hapten (a) comprising a first hapten and a labelling agentbound to said first hapten and an antibody (b) prepared by using animmunogen comprising a second hapten and an immunoactive carrier boundto said second hapten; and "case 2" being a combination of a firsthapten (c) and a labelled antibody (d) comprising an antibody (b)prepared by using an immunogen comprising a second hapten and an animmunoactive carrier bound to said second hapten and a labelling agentbound to said antibody.

In the "case 1", since the antibody (b) and the labelled hapten (a) inthe reagent are capable of undergoing reversible binding, a hapten in asample to be measured and the labelled hapten (a) in the reagent bindcompetitively to the antibody (b) in the reagent. In a device for use inan immunoassay according to the present invention, wherein such areagent is included in a single container, a hapten in a sample to bemeasured and the labelled hapten (a) bind competitively to the antibody(b) which is included in the container in an insolubilized form.

In the "case 2", since the hapten (c) and the labelled antibody (d) inthe reagent are capable of undergoing reversible binding, a hapten in asample to be measured and the hapten (c) in the reagent bindcompetitively to the labelled antibody (d) in the reagent. In a devicefor use in an immunoassay according to the present invention, whereinsuch a reagent is included in a single container, a hapten in a sampleto be measured and the hapten (c) which is included in the container inan insolubilized form bind competitively to the labelled antibody (d).

In either case, the amount of a hapten in a sample is measured bydetecting signals originating from a labelled insoluble substance afterB/F separation (B, bound form; F, free form).

When a substance to be measured is an antigen, the measuring principleis the same as in the case of a hapten as described above, except thatan immunoactive carrier is not necessary, and also provided that theterm "hapten" in the description is replaced by another term "antigen".The principle in the case of the measurement of a compound comprising ahapten and an immunoactive carrier or an antibody prepared by using anantigen as the immunogen can be explained in the same manner.

Briefly, the measuring device of the present invention is divided intotwo cases: a case in which a hapten (or an antigen) is included in acontainer in an insolubilized form, and an antibody in a sample and alabelled antibody bind competitively to the insoluble hapten (orantigen); and the other case in which an antibody is included in acontainer in an insolubilized form, and the insoluble antibody and anantibody in a sample bind competitively to a labelled hapten (or alabelled antigen comprising an antigen and a labelling agent).

In either case, the amount of an antibody in a sample is measured bydetecting signals originating from a labelled insoluble substance afterB/F separation.

Thus, the principle of the immunoassay making use of the immunoassayreagent of the present invention is explained hereinabove.

Next, a combination of a labelled hapten (a) and an antibody (b) andanother combination of a labelled antigen and an antibody are explained,which are applied to the reagent of the present invention for use insuch immunoassays.

It is preferred that first hapten of the labelled hapten (a) and thesecond hapten (which is used after binding it to an immunoactivecarrier) used as the immunogen for the preparation of the antibody (b)are analogous but different from each other, having different chemicalstructures, respectively. Since the binding ability between the labelledhapten (a) and the antibody (b) varies widely by the use of suchanalogous substances, a combination of a member of the group of thelabelled hapten (a) and a member of the group of the antibody (b), whichare capable of undergoing reversible binding, can be selected. In otherwords, the reaction between the labelled hapten (a) and the antibody (b)becomes reversible, if a hapten, whose chemical structure is analogousto but different from the second hapten used as the immunogen for thepreparation of the antibody (b) and whose cross-reaction ability withthe antibody is relatively small, is labelled and used as the labelledhapten (a). However, even if a first hapten for use in the labelledhapten (a) and a second hapten to be used as the immunogen for thepreparation of the antibody (b) have the same chemical structure, thesehaptens may also be applicable, because the labelled hapten (a) and theantibody (b) prepared by using these first and second haptens having thesame chemical structure are sometimes capable of undergoing reversiblebinding.

In a practical immunoassay, a substance to be measured becomes a thirdfactor of the chemical structures. In general, when the substance to bemeasured is a hapten, the second hapten used as the immunogen for thepreparation of the antibody (b) may have the same chemical structure asthat of the hapten to be measured or have a slightly modified chemicalstructure of the hapten to be measured but having the same antigenicity,and an immunoactive carrier is linked to a site of the second haptenwhere the antigenicity of the second hapten may not be changed.

A labelled hapten, on the other hand, may be obtained by labelling asubstance (hapten) having a certain chemical structure which has anaffinity to a prepared antibody (b) and is capable of undergoingreversible binding to the antibody (b).

When the substance to be measured is an antibody which is made againstan immunogen comprising a hapten and an immunoactive carrier, it ispreferred that a first hapten having the same chemical structure as thatof the hapten which is used in the immunogen for the preparation of theantibody to be measured or having a slightly modified chemical structureof the hapten in the immunogen but having the same antigenicity be usedfor the preparation of the labelled hapten (a), and a second haptenhaving a slightly different chemical structure from that of the haptenused in the immunogen for the preparation of the antibody to be measuredbe bound to an immunoactive carrier and used as the immunogen for use inthe preparation of the antibody (b).

When the substance to be measured is an antigen or an antibody which ismade against an antigen, conditions for the selection of the chemicalstructure of the first and second haptens are the same as those in thecase of the above-described substances to be measured (a hapten and anantibody which is made against an immunogen comprising a hapten and animmunoactive carrier), except that immunoactive carriers are notrequired.

The following explains a combination of a labelled antibody (d) and ahapten (c) and another combination of a labelled antibody and anantigen, which are applied to the reagent of the present invention.

It is preferred that a first hapten (c) for use in the immunoassayreagent of the present invention and a second hapten (which is usedafter binding it to an immunoactive carrier) used as the immunogen forthe preparation of the labelled antibody (d) are analogous but differentfrom each other having different chemical structures, respectively.Since the binding ability between the hapten (c) and the labelledantibody (d) varies widely by the use of such analogous substances, acombination of a member of the group of the hapten (c) and a member ofthe group of the labelled antibody (d), which are capable of undergoingreversible binding, can be selected. However, even if a first hapten (c)for use in the immunoassay reagent of the present invention and a secondhapten to be used as the immunogen for the preparation of the labelledantibody (d) have the same chemical structure, these haptens may also beapplicable, because the hapten (c) and the labelled antibody (d)prepared by using these first and second haptens having the samechemical structure are sometimes capable of undergoing reversiblebinding.

In general, when the substance to be measured is an antibody which ismade against an immunogen comprising a hapten and an immunoactivecarrier, a first hapten having the same chemical structure as that ofthe hapten which is used in the immunogen for the preparation of theantibody to be measured or having a slightly modified chemical structureof the hapten in the immunogen but having the same antigenicity is usedfor the preparation of the hapten (c), and a second hapten having aslightly modified chemical structure from that of the hapten used in theimmunogen for the preparation of the antibody to be measured is bound toan immunoactive carrier and used as the immunogen for use in thepreparation of the labelled antibody (d), because chemical structures ofthe first and second haptens are such that the hapten (c) for use in theimmunoassay reagent of the present invention and the labelled antibody(d) become capable of undergoing reversible binding.

When the substance to be measured is a hapten, the second hapten used asthe immunogen for the preparation of the labelled antibody (d) may havethe same chemical structure as that of the hapten to be measured or havea slightly modified chemical structure of the hapten to be measured buthaving the same antigenicity, and an immunoactive carrier is linked to asite of the second hapten where the antigenicity of the second haptenmay not be changed. The hapten (c) for use in the immunoassay reagent ofthe present invention, on the other hand, may be obtained by selecting acertain chemical structure which has an affinity to a labelled antibody(d) and is capable of undergoing reversible binding to the labelledantibody (d).

When the substance to be measured is an antigen, or the immunogen foruse in the preparation of an antibody to be measured is not a hapten butan antigen, conditions for the selection of the chemical structure ofthe first and second haptens are the same as those in the abovedescription wherein a substance to be measured or an immunogen for usein the preparation of an antibody to be measured is a hapten, exceptthat immunoactive carriers are not required.

The term "modified chemical structure" or the like as used hereinaboveis intended to include any derivative which can be prepared by usingcommonly used techniques.

Principles of the combination of a labelled hapten or a labelled antigenand an antibody and another combination of a hapten or an antigen and alabelled antibody, which are used in the measuring device of the presentinvention, are explained above. The following concretely describes theexample of samples which can be detected and measured by using theimmunoassay reagent or the immunoassay device of the present invention,and substances for use in the immunoassay reagent of the presentinvention for measuring these test samples.

The immunoassay reagent or the immunoassay device of the presentinvention can be used for the measurement of substances havingrelatively low molecular weights, which are called haptens, such as:steroids, which comprises estrogens including estrone, estradiol andestriol, gestagens including progesterone, androgens includingtestosterone, mineral corticoids including aldosterone, andglucocorticoids including corticosterone; thyroid hormones, includingthyroxine; amino acids and amines; drugs, including digoxin; andpeptides, including angiotensin and α-hANP; as well as metabolites ofthese substances.

The immunoassay reagent or the immunoassay device of the presentinvention can also be used for the measurement of antigens, such asluteinizing hormone (LH), thyroid-stimulating hormone (TSH), humanchronic gonadotropin (hCG) and carcinoembryonic antigen (CEA), as wellas metabolites thereof.

Still more, the immunoassay reagent or the immunoassay device of thepresent invention can be used for the measurement of antibodies whichare made in the body against immunogens, such as hepatitis B virus,rubella virus and various allergens.

The immunoassay reagent or the immunoassay device of the presentinvention is useful especially for the measurement of haptens, moreespecially for the measurement of estrogens and their metabolites andgestagens and their metabolites. The following exemplifies thecombination of antibodies and labelled haptens for use in theimmunoassay reagent and the immunoassay device of the present inventionfor the measurement of urinary estrogen, blood estradiol or bloodprogesterone.

For the measurement of urinary estrogen or blood estradiol using theimmunoassay reagent or the immunoassay device of the present invention,it is preferred to use a labelled hapten (a) in which a first hapten forthe preparation of the labelled hapten (a) is selected from the groupconsisting of compounds represented by the following formulae I to IVand an antibody (b) which is prepared by using an immunogen comprising asecond hapten selected from the group consisting of compoundsrepresented by the following formulae I to IV and an immunoactivecarrier bound to the second hapten: ##STR1## wherein R¹ is hydrogen, andR² is Y¹ --Z; or R¹ is Y² --Z and R² is oxygen;

wherein Y¹ is a straight-chain, branched, or cyclic radical interveningZ and the steroid nucleus comprising a backbone having 1 to 10 carbonatoms and/or hetero atoms, bonded to the steroid nucleus by a doublebond; Y² is a straight-chain, branched, or cyclic radical intervening Zand the oxygen comprising a backbone having 0 to 10 carbon atoms and/orhetero atoms bonded to the oxygen by a single bond; and Z is a radicalselected from the group consisting of hydrogen, --NH₂, --SH, --COOH,--CHO and ##STR2## wherein R³, R⁴ and R⁵ are hydrogen, one of R⁶ and R⁷is hydrogen, and the other is Y³ --Z; R³ is Y² --Z, R⁴, R⁵ and R⁷ arehydrogen, and R⁶ is Y³ --Z; R³ and R⁷ are hydrogen, one of R⁴ and R⁵ ishydrogen and the other is Y³ --Z, and R⁶ is hydroxyl; or R³ and R⁷ arehydrogen, R⁴ and R⁵ together represent Y¹ --Z bonded to the steroidnucleus by a double bond, and R⁶ is hydroxyl;

wherein Y¹ is a straight-chain, branched, or cyclic radical interveningZ and the steroid nucleus comprising a backbone having 1 to 10 carbonatoms and/or hetero atoms, bonded to the steroid nucleus by a doublebond; Y² is a straight-chain, branched, or cyclic radical intervening Zand the oxygen comprising a backbone having 0 to 10 carbon atoms and/orhetero atoms bonded to the oxygen by a single bond; Y³ is astraight-chain, branched, or cyclic radical intervening Z and thesteroid nucleus comprising a backbone having 1 to 10 carbon atoms and/orhetero atoms, bonded to the steroid nucleus by a single bond; and Z is aradical selected from the group consisting of hydrogen, --NH₂, --SH,--COOH, --CHO and ##STR3## wherein R⁸ is hydrogen, R⁹ is Y³ --Z and oneof R¹⁰ and R¹¹ is Y³ --Z, and the other is hydrogen; R⁸ is hydrogen, oneof R⁹ and R¹⁰ is hydroxyl and the other is Y³ --Z, and R¹¹ is hydrogen;or R⁸ is Y² --Z, R⁹ is Y³ --Z, R¹⁰ is Y⁴ --Z, and R¹¹ is hydrogen; and

wherein Y² is a straight-chain, branched, or cyclic radical interveningZ and the oxygen comprising a backbone having 0 to 10 carbon atomsand/or hetero atoms bonded to the oxygen by a single bond; Y³ and Y⁴ areindividually a straight-chain, branched, or cyclic radical intervening Zand the steroid nucleus comprising a backbone having 1 to 10 carbonatoms and/or hetero atoms, bonded to the steroid nucleus by a singlebond; and Z is a radical selected from the group consisting of hydrogen,--NH₂, --SH, --COOH, --CHO and ##STR4## wherein one of R¹², R¹³, R¹⁴,R¹⁵, R¹⁶ and R¹⁷ is Y³ --Z and the others are hydrogen; or R¹² and R¹³,R¹⁴ and R¹⁵, or R¹⁶ and R¹⁷ together represent Y¹ --Z bonded to thesteroid nucleus by a double bond, and the others are hydrogen; whereinY¹ is a straight-chain, branched, or cyclic radical intervening Z andthe steroid nucleus comprising a backbone having 1 to 10 carbon atomsand/or hereto atoms, bonded to the steroid nucleus by a double bond; Y³is a straight-chain, branched, or cyclic radical intervening Z and thesteroid nucleus comprising a backbone having 1 to 10 carbon atoms and/orhereto atoms, bonded to the steroid nucleus by a single bond; and Z is aradical selected from the group consisting of hydrogen, --NH₂, --SH,--COOH, --CHO and ##STR5##

Examples of the compounds represented by the formulae I to IV are listedbelow.

The compound corresponding to the formula I includes estrone,estrone-17-carboxymethyl oxime and estrone-3-carboxymethyl ether. Thecompound corresponding to the formula II includes estradiol,estradiol-3-carboxymethyl ether, estradiol-6α-hemisuccinate,estradiol-6-carboxymethyl oxime, estradiol-17α-hemisuccinate andestradiol-17β-hemisuccinate. The compound corresponding to the formulaIII includes estriol, estriol-3-carboxymethyl ether, estriol-16α,17β-dihemisuccinate, estriol-16β, 17β-dihemisuccinate,estriol-16α-glucuronide and estriol-17β-glucuronide. The compoundcorresponding to the formula IV comprises estradiol-7-hemisuccinate,estradiol-7-carboxyethyl thioether, estradiol-11-hemisuccinate andestradiol-16α-hemisuccinate.

The following describes preparation methods and combinations ofantibodies and labelled haptens for use in the immunoassay reagent andthe immunoassay device of the present invention for measuring urinaryestrogen.

Measurement of urinary estrogen is usually applied to a monitor systemof the fetoplacental unit function in pregnant women, because conditionsof the fetoplacental unit in a pregnant woman can be evaluated bymeasuring the amount of estriol which is produced in the fetoplacentalunit and excreted into urine of the pregnant woman.

The major component of urinary estrogen comprises estriol derivatives,and estriol-16α-glucuronide is the most important derivative among them.For the measurement of urinary estrogen, therefore, it is desirable touse an antibody to estriol-16α-glucuronide as the main substance to bemeasured, which is cross-reactive to other coexisting conjugatedestriols, conjugated estradiols and conjugated estrones in the sample.

Such an antibody may be obtained by immunizing an animal with animmunogen which comprises an immunoactive carrier and an estriolderivative, an estradiol derivative or an estrone derivative and thenpreparing a hybridoma which produces an anti-estrogen antibody by meansof a cell fusion technique.

Since the main component of urinary estrogen is a conjugated estriol,the use of an estriol derivative, rather than an estrone derivative, iseffective as a hapten of the immunogen which is used for the preparationof an antibody for use in the measurement of urinary estrogen.

Antibodies thus prepared are then checked for their cross-reactivitiesand measurable ranges of estrogen, in order to select an antibody whichshows about 100 ng/ml of the 50%-inhibition concentration ofestriol-16α-glucuronide, estimated from a standard curve, and hascross-reactivities to estriol, estradiol, estrone, conjugated estriol atthe 17-position, conjugated estriol at the 16-position, conjugatedestriol at the 16- and 17-positions, conjugated estradiol at the17-position, and the like.

Preparation of a labelled estrogen is performed by selecting an estrogenderivative whose binding ability to the antibody is slightly lower thanor similar to that of the estrogen to be measured(estriol-16α-glucuronide). For this purpose, results of thecross-reactivity test of antibodies are used as a reference.

For example, if an antibody prepared by using an immunogen comprising anestriol-16,17-derivative and an immunoactive carrier bound to thederivative is used, a labelled estrogen corresponding to the antibodymay preferably be prepared using an estrone-17-derivative. Naturally,this combination of the antibody and the labelled estrogen is in no wayto be taken as limiting.

The following describes the preparation and combination of antibodiesand labelled haptens wherein the substance to be measured is bloodestradiol.

Measurement of blood estradiol is applied to a monitor system of thefemale gonadal functions, especially maturation of ovarian follicles andovulation.

The antibody for use in the measurement of estradiol may be obtained byimmunizing an animal with an immunogen which comprises an estradiol-3,6, 7, 11 or 16-derivative and an immunoactive carrier bound to thederivative and then preparing a hybridoma which produces ananti-estradiol antibody by means of a cell fusion technique.

An antibody prepared by using an immunogen which comprises anestradiol-3 or 6-derivative and an immunoactive carrier bound to thederivative is advantageous in view of the cross-reactivity.

Antibodies thus prepared are then checked for their measurable ranges ofestradiol and cross-reactivities to each of the steroids and theirderivatives, in order to select an antibody which shows about 200 pg/mlof the 50%-inhibition concentration of estradiol, estimated from astandard curve, and has small cross-reactivities to other steroids thanestradiol.

When an antibody for use in the measurement of blood estradiol isprepared by using an immunogen comprising an estradiol-6-derivative andan immunoactive carrier bound to the derivative, a labelled estradiolcorresponding to the antibody may preferably be prepared using anestradiol-3-derivative or even the same estradiol-6-derivative providedthat the derivatives has a different bridge structure. Naturally, thiscombination of the antibody and the labelled estradiol is in no way tobe taken as limiting.

The following describes the preparation and combination of antibodiesand labelled haptens wherein the substance to be measured is bloodprogesterone.

For the measurement of blood progesterone, it is preferred that theimmunoassay reagent or the immunoassay device of the present inventioncomprises (a) a labelled hapten comprising a first hapten and alabelling agent bound to the first hapten and (b) an antibody preparedby using an immunogen comprising a second hapten and an immunoactivecarrier bound to the second hapten, wherein the first and second haptensare individually selected from the group consisting of compoundsrepresented by the formula V: ##STR6## wherein R¹⁸ is oxygen, one ofR¹⁹, R²⁰, R²¹, R²² and R²³ is Y⁵ --Z, and others are hydrogen; or R¹⁸ isY¹ --Z and R¹⁹, R²⁰, R²¹, R²² and R²³ are hydrogen; wherein Y¹ is astraight-chain, branched, or cyclic radical intervening Z and thesteroid nucleus comprising a backbone having 1 to 10 carbon atoms and/orhereto atoms, bonded to the steroid nucleus by a double bond, Y⁵ is astraight-chain, branched, or cyclic radical intervening Z and thesteroid nucleus or the methylene radical bonded to the steroid nucleuscomprising a backbone having 1 to 10 carbon atoms and/or hereto atoms,bonded to the steroid nucleus or the methylene radical bonded to thesteroid nucleus by a single bond, and Z is a radical selected from thegroup consisting of hydrogen, --NH₂, --SH, --COOH, --CHO and ##STR7##

The compound represented by the formula V includes progesterone,progesterone-3-carboxymethyl oxime, progesterone-11α-hemisuccinate,progesterone-16α-hemisuccinate, progesterone-17α-hemisuccinate,progesterone-18-hemisuccinate and progesterone-19-hemisuccinate.

The following describes preparation methods and combinations ofantibodies and labelled haptens for use in the immunoassay reagent andthe immunoassay device of the present invention for measuring bloodprogesterone.

Measurement of blood progesterone is applied to the diagnosis of women'sgonadal functions, especially luteal function.

The antibody for use in the measurement of progesterone may be obtainedby immunizing an animal with an immunogen which comprises aprogesterone-3, 11, 16, 17, 18 or 19-derivative and an immunoactivecarrier bound to the derivative and then preparing a hybridoma whichproduces an anti-progesterone antibody by means of a cell fusiontechnique.

An antibody prepared by using an immunogen which comprises anprogesterone-11-derivative and an immunoactive carrier bound to thederivative is advantageous in view of the cross-reactivity.

Antibodies thus prepared are then checked for their measurable ranges ofprogesterone and cross-reactivities to each of the steroids and theirderivatives, in order to select an antibody which shows about 3 ng/ml ofthe 50%-inhibition concentration of progesterone, estimated from astandard curve, and has small cross-reactivities to steroids other thanprogesterone.

When an antibody for use in the measurement of blood progesterone isprepared by using an immunogen comprising an progesterone-11-derivativeand an immunoactive carrier bound to the derivative, a labelledprogesterone corresponding to the antibody may preferably be preparedusing a progesterone-19-derivative. Naturally, this combination of theantibody and the labelled progesterone is in no way to be taken aslimiting.

Any one of bovine serum albumin, keyhole limpet hemocyanin,Thyroglobulin and the like may be used as an immunoactive carrier in animmunogen for use in the preparation of an antibody which is prepared byusing the immunogen comprising a second hapten and the immunoactivecarrier bound to the second hapten. Binding of an immunoactive carrierto a hapten may be performed in accordance with any known methoddescribed in textbooks and the like, such as Koso Men-eki Sokuteiho[(Enzyme Immunoassay, written in Japanese); published by Igaku ShoinCo., Ltd.; p. 46-60] and Zoku Rajio imuno assei [(Radioimmunoassay,second series; written in Japanese); published by Kodansha Co., Ltd.; p.56-62 and P. 78-87].

Either monoclonal or polyclonal antibodies may be useful as the antibodyof the present invention, but, if a polyclonal antibody is used, it ispreferable that the polyclonal antibody be purified into a fractionsuitable for the measuring purpose by means of affinity chromatographyor the like. Therefore, monoclonal antibodies are preferable as theantibody of the present invention from a workability point of view.Animals for the preparation of antibodies are not limited, but mice aregenerally used.

With regard to the labelling agent, any of the commonly used agents,such as enzymes including horseradish peroxidase and alkalinephosphatase, radioisotopes including ¹²⁵ I, fluorescent materialsincluding fluorescein, a fluorescein derivative and europium, andchemiluminescent materials including an acridinium derivative, may beused, preferably an enzyme from the viewpoint of easy handling.

Binding of the labelling agent to a hapten, an antigen or an antibodymay be performed in accordance with any known method described intextbooks and the like, such as Koso Men-eki Sokuteiho [(EnzymeImmunoassay, written in Japanese); published by Igaku Shoin Co., Ltd.;p. 46-60].

The immunoassay device of the present invention, wherein the immunoassayreagent of the present invention (a combination of a labelled hapten andan antibody, a labelled antigen and an antibody, a hapten and a labelledantibody or an antigen and a labelled antibody) is included in acontainer made of a plastic material, a glass material or the like, isproduced as follows.

Firstly, an antibody, a hapten or an antigen to be insolubilized isincluded in a container and insolubilized by means of chemical covalentbonding or physical adsorption. To this is added a portion of a buffersolution containing a corresponding labelled hapten, labelled antigen orlabelled antibody, and the mixture is then incubated for a period a timewhich is sufficient for the completion or near-completion of attainingthe equilibrium state of the immune reaction. Thereafter, the contentsin the container are subjected to freezing or freeze-drying.

The measuring process is started by the injection of a sample into thecontainer thus prepared. After a certain period of incubation of thesample-added container (a time which is sufficient for attainingcompletion or near-completion of the equilibrium state of the immunereaction), a B/F separation is performed, and the amount of thesubstance to be measured in the sample is calculated on the basis of asignal (activity or physical value) originating from the remainedlabelled material after the B/F separation. The presence of thesubstance to be detected can be judged in the same manner.

The labelled hapten or the labelled antigen (or the labelled antibody)and the corresponding antibody (or hapten or antigen) in the reagent ofthe present invention are capable of undergoing reversible binding. Whena sample is added to the reagent, therefore, a part of the labelledhapten or labelled antigen (or labelled antibody) once bound to itscorresponding antibody (or hapten or antigen) is unbound again inproportion to the amount of a substance to be measured in the sampleand, instead, the substance to be measured in the sample is bound to itscorresponding antibody, hapten or antigen. In consequence, in spite ofthe previous binding of the labelled substance to its correspondingantibody, hapten or antigen an by immune reaction, a competitive bindingreaction starts between the labelled substance and a substance to bemeasured when the sample is added to the reagent afterward and thereaction reaches a certain equilibrium state, thus rendering possibledevelopment of the competitive binding immunoassay system of the presentinvention.

When the immunoassay reagent or the immunoassay device of the presentinvention is used, the equilibrium state of the immune reaction can beobtained quickly within about 30 minutes.

EXAMPLES

Examples of the present invention are given below by way ofillustration, and not by way of limitation.

Example 1

Preparation of estradiol-17epi-hemisuccinate,estriol-16,17-dihemisuccinate, estriol-16epi, 17-dihemisuccinate,progesterone-19-hemisuccinate, progesterone-16α-hemisuccinate,progesterone-17α-hemisuccinate and progesterone-18-hemisuccinate.

Each of 17epi-estradiol, estriol, 16epi-estriol, 19-hydroxyprogesterone, 16α-hydroxy progesterone, 17α-hydroxy progesterone, and18-hydroxy progesterone (all purchased from Sigma Chemical Co.) wasdissolved in pyridine, mixed with succinic anhydride (10 to 30 times asmuch as the starting material on molar basis), and then subjected toheating reflux under nitrogen atmosphere. After cooling the resultingreaction mixture by adding an appropriate amount of water, the waterphase was neutralized with dilute hydrochloric acid, and the product wasextracted from the water phase by ethyl acetate. The resulting ethylacetate phase was washed with dilute hydrochloric acid, water, andsaturated sodium chloride solution, in that order.

For the isolation of estrogen derivatives, the acid fraction wasextracted and separated from the ethyl acetate phase using a saturatedsodium carbonate solution. To the acid fraction cooled in an ice bathwas added dilute hydrochloric acid in order to acidify the aqueoussolution, and the product was extracted by ethyl acetate. The ethylacetate phase was washed with water and saturated sodium chloridesolution, in that order.

Anhydrous sodium sulfate was added to the ethyl acetate solution. Afterdrying the mixture, ethyl acetate remaining in the dried material wasevaporated under reduced pressure. Each steroid derivative thus obtainedwas checked for its purity by thin layer chromatography, and stored forlater use. When contamination of the starting material was found by thinlayer chromatography, the product was further separated and purified bysilica gel column chromatography.

Conditions for the synthesis of each derivative and its yield are shownin Table 1.

                  TABLE 1                                                         ______________________________________                                                        Succinic Reflux                                                               anhydride                                                                              time    Yield                                                        (molar ratio)                                                                          (hr)    (%)                                          ______________________________________                                        Estradiol-17epi-hemisuccinate                                                                   20         18       3                                       Estriol-16,17-dihemisuccinate                                                                   30         20      30                                       Estriol-16epi,17-dihemisuccinate                                                                30         15       5                                       Progesterone-19-hemisuccinate                                                                   10         3       80                                       Progesterone-16α-hemisuccinate                                                            10         5       60                                       Progesterone-17α-hemisuccinate                                                            10         3       55                                       Progesterone-18-hemisuccinate                                                                   10         3       60                                       ______________________________________                                    

Example 2

Preparation of estrone-17-carboxymethyl oxime.bovine serum albumin,estradiol-17-hemisuccinate.bovine serum albumin,estriol-16,17-dihemisuccinate.bovine serum albumin,estriol-16α-glucuronide.bovine serum albumin, estradiol-3-carboxymethylether.bovine serum albumin, estradiol-6-carboxymethyl oxime.bovine serumalbumin, estradiol-6α-hemisuccinate.bovine serum albumin,progesterone-11α-hemisuccinate.bovine serum albumin,progesterone-19-hemisuccinate.bovine serum albumin,progesterone-16α-hemisuccinate.bovine serum albumin,progesterone-17α-hemisuccinate.bovine serum albumin,progesterone-18-hemisuccinate.bovine serum albumin, andprogesterone-3-carboxymethyl oxime.bovine serum albumin.

Bovine serum albumin was bound to each of the steroid derivatives listedbelow by means of a mixed acid anhydride method:

Steroid derivatives prepared in Example 1 consisting ofestriol-16,17-dihemisuccinate, progesterone-19-hemisuccinate,progesterone-16α-hemisuccinate, progesterone-17α-hemisuccinate andprogesterone-18-hemisuccinate; steroid derivatives purchased from SigmaChemical Co. consisting of estrone-17-carboxymethyl oxime,estradiol-17-hemisuccinate, estriol-16α-glucuronide,estradiol-6-carboxymethyl oxime, progesterone-11α-hemisuccinate andprogesterone-3-carboxymethyl oxime; and steroid derivatives purchasedfrom Mitani Sangyo Co., Ltd. consisting of estradiol-3-carboxymethylether and estradiol-6α-hemisuccinate.

The mixed acid anhydride of each steroid derivative was prepared bydissolving each steroid derivative in dioxane and incubating thesolution at 12°±2° C. for 20 minutes with gradual dripping ofchloroisobutyl formate (equivalent amount to the steroid derivative on amolar basis) in the presence of tri-n-butylamine (molar ratio to thederivative, 1.1).

The mixed acid anhydride of each steroid derivative thus obtained wasimmediately bound to bovine serum albumin in the following manner.Bovine serum albumin was dissolved in a 50% aqueous solution of dioxaneto a final concentration of 10 mg/ml, and the albumin solution wasadjusted to pH 8. After cooling down the albumin solution to 10°±2° C.,the mixed acid anhydride of each steroid derivative previously preparedwas dripped into the albumin solution, with the molar ratio of theanhydride to the bovine serum albumin being 5 to 100. After completionof the dripping, the resulting solution was adjusted to pH 8 and stirredfor another 2 hours at 10°±2° C. Thereafter, the reaction solution wastransferred into a semi-permeable membrane tube and dialyzed against 100volumes of physiological saline. The dialysis process was repeated threetimes and then the product was purified by means of Sephadex G-50 columnchromatography.

Example 3

Preparation of estrone-17-carboxymethyl oxime.peroxidase,estradiol-17-hemisuccinate.peroxidase,estriol-16,17-dihemisuccinate.peroxidase,estriol-16α-glucuronide.peroxidase, estradiol-17epihemisuccinate.peroxidase, estriol-16epi, 17-dihemisuccinate.peroxidase,estradiol-3-carboxymethyl ether.peroxidase, estradiol-6-carboxymethyloxime.peroxidase, estradiol-6α-hemisuccinate.peroxidase,progesterone-11α-hemisuccinate.peroxidase,progesterone-19-hemisuccinate.peroxidase,progesterone-16α-hemisuccinate.peroxidase andprogesterone-3-carboxymethyl oxime.peroxidase.

Horseradish peroxidase was purchased from Toyobo Co., Ltd. and bound toeach of the steroid derivatives listed below by means of a mixed acidanhydride method or an N-hydroxysuccinimide ester activation method:

Steroid derivatives purchased from Sigma Chemical Co. consisting ofestrone-17-carboxymethyl oxime, estradiol-17-hemisuccinate,estriol-16α-glucuronide, estradiol-6-carboxymethyl oxime,progesterone-11α-hemisuccinate and progesterone-3-carboxymethyl oxime;steroid derivatives purchased from Mitani Sangyo Co., Ltd. consisting ofestradiol-3-carboxymethyl ether and estradiol-6α-hemisuccinate; andsteroid derivatives prepared in Example 1 consisting ofestriol-16,17-dihemisuccinate, estradiol-17epihemisuccinate,estriol-16epi,17-dihemisuccinate, progesterone-19-hemisuccinate andprogesterone-16α-hemisuccinate.

(3-1) Mixed acid anhydride method.

The mixed acid anhydride of each steroid derivative was prepared bydissolving each steroid derivative in dioxane and incubating thesolution at 12°±2° C. for 20 minutes with gradual dripping ofchloroisobutyl formate (equivalent amount to the steroid derivative on amolar basis) in the presence of tri-n-butylamine (molar ratio to thederivative, 1.1).

The mixed acid anhydride of each steroid derivative thus obtained wasimmediately bound to horseradish peroxidase in the following manner.Horseradish peroxidase was dissolved in a 50% aqueous solution ofdioxane to its final concentration of 5 mg/ml, and the horseradishperoxidase solution was adjusted to pH 8. After cooling down theperoxidase solution to 10°±2° C., the mixed acid anhydride of eachsteroid derivative previously prepared was dripped into the peroxidasesolution, with molar ratio of the anhydride to the horseradishperoxidase being 1 to 100. After completion of the dripping, theresulting solution was adjusted to pH 8 and stirred for another 2 hoursat 10°±2° C. Thereafter, the reaction solution was transferred into asemi-permeable membrane tube and dialyzed against 100 volumes of 0.076Mphosphate buffered saline (pH 7.0). The dialysis process was repeatedthree times and then the product was purified by means of Sephadex G-50column chromatography.

(3-2) N-hydroxysuccinimide ester activation method.

Each steroid derivative was dissolved in a 95% dioxane aqueous solution,and the resulting solution was mixed with N-hydroxysuccinimide (molarratio to the steroid derivative, 1.5) and water-soluble carbodiimide(molar ratio, 2) and stirred for 1 to 6 hours. Ethyl acetate was addedto the resulting reaction solution and the product was extracted fromthe water phase by an ethyl acetate phase. The ethyl acetate phase waswashed with water and saturated sodium chloride, solution in that order.

Anhydrous sodium sulfate was added to the ethyl acetate solution. Afterdrying the mixture, ethyl acetate remaining in the dried material wasevaporated under reduced pressure. The activated steroid derivative thusobtained was dissolved in dioxane and stored at 0° C. or below.

Horseradish peroxidase was dissolved in 50 mM phosphate buffer solution(pH 7.6) to its final concentration of 10 mg/ml. After cooling down theperoxidase solution to 4°±2° C., the activated steroid derivativepreviously prepared was dripped into the peroxidase solution, with themolar ratio of the activated steroid derivative to the horseradishperoxidase being 1 to 50. After completion of the dripping, theresulting solution was stirred for another 4 hours at 4°±2° C.Thereafter, the reaction solution was transferred into a semi-permeablemembrane tube and dialyzed against 100 volumes of 0.076M phosphatebuffered saline (pH 7.0). The dialysis process was repeated three timesand then the product was purified by means of Sephadex G-50 columnchromatography.

Conditions for the synthesis of each labelled steroid derivative areshown in Table 2.

                  TABLE 2                                                         ______________________________________                                                                        Molar                                         Peroxidase-labelled    Labelling                                                                              ratio of                                      steroid derivative     method*  steroid                                       ______________________________________                                        Estrone-17-carboxymethyl oxime.peroxidase                                                            A        1 to 10                                       Estradiol-17-hemisuccinate.peroxidase                                                                A        1 to 30                                       Estriol-16,17-dihemisuccinate.peroxidase                                                             A        1 to 10                                       Estriol-16α-glucuronide.peroxidase                                                             A        5 to 50                                       Estradiol-17epi-hemisuccinate.peroxidase                                                             A        10 to 100                                     Estriol-16epi,17-dihemisuccinate.peroxidase                                                          A        10 to 100                                     Estradiol-3-carboxymethyl ether.peroxidase                                                           A        1 to 30                                       Estradiol-6-carboxymethyl oxime.peroxidase                                                           A        1 to 30                                       Estradiol-6α-hemisuccinate.peroxidase                                                          B        1 to 10                                       Progesterone-11α-hemisuccinate.peroxidase                                                      B        1 to 10                                       Progesterone-19-hemisuccinate.peroxidase                                                             B        1 to 30                                       Progesterone-16α-hemisuccinate.peroxidase                                                      B        1 to 50                                       Progesterone-3-carboxymethyl                                                                         A        1 to 30                                       oxime.peroxidase                                                              ______________________________________                                         *:A, Mixed acid anhydride method; and B, Nhydroxysuccinimide ester            activation method.                                                       

Example 4

Preparation of monoclonal anti-estrogen antibodies.

Balb/c mice were immunized with an immunogen prepared by binding anestrogen derivative to bovine serum albumin. Changes in the bloodantibody titer were monitored while boostering was performed, and spleencells of a mouse in which the antibody titer was increased were used forcell fusion. Cell fusion was performed in accordance with the methoddescribed in Methods in Enzymology (vol. 73, p. 3 to 46). Culturesupernatants of hybridomas thus obtained were screened for theanti-estrogen antibody, and positive hybridomas were cloned. Antibodiesthus selected were further checked for their cross-reactions andmeasurable ranges of estrogen, in order to eliminate any antibody whichwas found to be inappropriate for the measuring of urinary estrogen. Anappropriate antibody produced in a culture supernatant of a hybridomathus selected was purified by means of affinity column chromatographyusing protein A.

The process for the preparation of hybridomas which produceanti-estrogen antibodies is shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                          No. of                                                                        immu-    No. of  No. of                                                       nized    cell    antibodies                                 Immunogen         mice     fusions selected                                   ______________________________________                                        Estrone-17-carboxymethyl oxime.                                                                 10       2        0                                         bovine serum albumin                                                          Estradiol-17-hemisuccinate.                                                                     10       8       30                                         bovine serum albumin                                                          Estriol-16,17-dihemisuccinate.                                                                  10       9       65                                         bovine serum albumin                                                          Estriol-16α-glucuronide.                                                                  10       1        0                                         bovine serum albumin                                                          ______________________________________                                    

Example 5

Preparation of reagent for use in the measurement of urinary estrogen.

One of the purified monoclonal anti-estrogen antibodies was immobilizedin a glass test tube. A portion of a solution containing one of theestrogen derivatives labelled with horseradish peroxidase was added tothe test tube, and the labelled antigen was allowed to react with theinsoluble antibody for 60 minutes. A portion of a standard estrogensolution (10, 50, 200 or 1000 ng/ml) was then added to the reactionsystem and the immune reaction was performed for 20 minutes, followed bya washing process. Thereafter, the enzyme (peroxidase) reaction wasperformed for 10 minutes in the presence of hydrogen peroxide as thesubstrate and orthophenylenediamine as the color reagent, and then theabsorbance of the colored solution was measured.

The solution containing the peroxidase-labelled estrogen derivative wasadded to another glass tube in which the purified monoclonalanti-estrogen antibody was immobilized. A standard estrogen solution wasadded immediately after the addition of the solution of theperoxidase-labelled estrogen derivative. After performing 20 minutes ofthe immune reaction, the enzyme reaction was performed in the samemanner as described above, and the result was compared to that of theabove result.

Examples of the results are shown in FIGS. 1a, 1b, 1c and 1d, whereinE15-008 (immunogen, estriol-16,17-dihemisuccinate.bovine serum albumin)or E17-102 (immunogen, estradiol-17-hemisuccinate.bovine serum albumin)was used as the antibody and estrone-17-carboxymethyl oxime.peroxidaseor estriol-16,17-dihemisuccinate.peroxidase was used as the labelledestrogen derivative.

It was found that a combination of the antibody E15-008 and theperoxidase-labelled estrone-17-carboxymethyl oxime was appropriate forthe measurement of urinary estrogen. Also found that E5-057 (immunogen,estriol-16,17-dihemisuccinate.bovine serum albumin) and E7-006(immunogen, estradiol-17-hemisuccinate.bovine serum albumin) wereappropriate for the measurement of urinary estrogen (data not shown).

FIG. 2 shows changes in the immune reaction during the reaction periodafter the addition of a standard estrogen solution, in the case of acombination of the antibody E15-008 and the peroxidase-labelledestrone-17-carboxymethyl oxime.

Based on the result shown in FIG. 2, it was confirmed that the reactionof the antibody with the peroxidase-labelled estrogen is reversible, andthe immune reaction, in which the estrogen to be measured in the sample(standard estrogen solution) and the labelled estrogen derivative bindcompetitively to the antibody, reaches its equilibrium state quickly(about 10 minutes).

Example 6

Preparation of monoclonal anti-estradiol antibodies.

Mice were immunized with an immunogen prepared by binding an estradiolderivative to bovine serum albumin. Changes in the blood antibody titerwere monitored while boostering was performed, and spleen cells of amouse in which the antibody titer was increased were used for cellfusion. Cell fusion was performed in accordance with the methoddescribed in Methods in Enzymology (vol. 73, p. 3 to 46). Culturesupernatants of hybridomas thus obtained were screened for theanti-estradiol antibody, and positive hybridomas were cloned. Antibodiesthus selected were further checked for their cross-reactions andmeasurable ranges of estradiol, in order to eliminate any antibody whichwas found to be inappropriate for the measuring of blood estradiol. Anappropriate antibody produced in a culture supernatants of a hybridomathus selected was purified by means of affinity column chromatographyusing protein A.

The process for the preparation of hybridomas which produceanti-estradiol antibodies is shown in Table 4.

                  TABLE 4                                                         ______________________________________                                                                               No. of                                                          No. of        anti-                                                  Immu-    immu-   No. of                                                                              bodies                                                 nized    nized   cell  se-                                    Immunogens      mice     mice    fusions                                                                             lected                                 ______________________________________                                        Estradiol-3-carboxymethyl                                                                     Balb/c   10       4    0                                      ether.bovine serum albumin                                                                    ddY      10       3    1                                      Estradiol-6-carboxymethyl                                                                     Balb/c   30      14    11                                     oxime.bovine serum albumin                                                                    ddY      20      11    12                                     Estradiol-6α-hemisuccinate.                                                             Balb/c   30       9    3                                      bovine serum albumin                                                                          ddY      30      14    4                                      ______________________________________                                    

Example 7

Preparation of reagent for use in the measurement of blood estradiol.

One of the purified monoclonal anti-estradiol antibodies was immobilizedin a glass test tube. To this was added a portion of a solutioncontaining one of the estradiol derivatives labelled with horseradishperoxidase, and the labelled antigen was allowed to react with theinsoluble antibody for 60 minutes. A portion of a standard estradiolsolution (10, 50, 200, 1000 or 5000 pg/ml) was then added to thereaction system and the immune reaction was performed for 20 minutes,followed by a washing process. Thereafter, the enzyme (peroxidase)reaction was performed for 10 minutes in the presence of hydrogenperoxide as the substrate and orthophenylenediamine as the colorreagent, and then the absorbance of the colored substance was measured.

To another glass tube in which the purified monoclonal anti-estradiolantibody had been immobilized was added the solution containing theperoxidase-labelled estradiol derivative, followed by the immediateaddition of the standard estradiol solution by eliminating the step forthe reaction of the insoluble antibody with the labelled antigen. Afterperforming 20 minutes of the immune reaction, the enzyme reaction wasperformed in the same manner as described above, and the result wascompared to that of the above result.

Examples of the results are shown in FIGS. 3a, 3b, 3c and 3d, wherein E₂13-074 (immunogen, estradiol-6-carboxymethyl oxime.bovine serum albumin)or E₂ 26-109 (immunogen, estradiol-6-carboxymethyl oxime.bovine serumalbumin) was used as the antibody and estradiol-6-carboxymethyloxime-peroxidase or estradiol-6α-hemisuccinate.peroxidase was used asthe labelled estradiol derivative.

It was found that a combination of the antibody E₂ 13-074 and theperoxidase-labelled estradiol-6α-hemisuccinate was appropriate for themeasurement of blood estradiol. Antibodies other than the E₂ 13-074 werefound to be inappropriate for the measurement of blood estradiol.

FIG. 4 shows changes in the immune reaction during the reaction periodafter the addition of a standard estradiol solution, in the case of acombination of the antibody E₂ 13-074 and the peroxidase-labelledestradiol-6α-hemisuccinate.

Based on the result as shown in FIG. 4, it was confirmed that thereaction of the antibody with the peroxidase-labelled estradiol isreversible, and the immune reaction, in which the estradiol to bemeasured in the sample (standard estradiol solution) and the labelledestradiol derivative bind competitively to the antibody, reaches itsequilibrium state quickly (about 20 minutes).

Example 8

Preparation of monoclonal anti-progesterone antibodies.

Mice were immunized with an immunogen prepared by binding a progesteronederivative to bovine serum albumin. Changes in the blood antibody titerwere monitored while boostering was performed, and spleen cells of amouse in which the antibody titer was increased were used for cellfusion. The cell fusion was performed in accordance with the methoddescribed in Methods in Enzymology (vol. 73, p. 3 to 46). Culturesupernatants of hybridomas thus obtained were screened for theanti-progesterone antibody, and positive hybridomas were cloned.Antibodies thus selected were further checked for their cross-reactionsand measurable ranges of progesterone, in order to eliminate anyantibody which was found to be inappropriate for the measuring of bloodprogesterone. An appropriate antibody produced in a culture filtrate ofa hybridoma thus selected was purified by means of affinity columnchromatography using protein A.

The process for the preparation of hybridomas which produceanti-progesterone antibodies is shown in Table 5.

                  TABLE 5                                                         ______________________________________                                                              No. of                                                               Immu-    immu-    No. of                                                                              No. of                                                nized    nized    cell  antibodies                               Immunogens   mice     mice     fusions                                                                             selected                                 ______________________________________                                        Progesterone-11α-                                                                    Balb/c   10       8     9                                        hemisuccinate.bovine                                                          serum albumin                                                                              ddY      10       3     11                                       Progesterone-19-                                                                           Balb/c   10       4     4                                        hemisuccinate.bovine                                                          serum albumin                                                                              ddY      10       3     2                                        Progesterone-16α-.                                                                   Balb/c   10       3     0                                        hemisuccinate.bovine                                                          serum albumin                                                                              ddY       5       2     0                                        Progesterone-3-                                                                            Balb/c    5       1     0                                        carboxymethyl oxime.                                                          bovine serum albumin                                                          ______________________________________                                    

Example 9

Preparation of a reagent for use in the measurement of bloodprogesterone.

One of the purified monoclonal anti-progesterone antibodies wasimmobilized in a glass test tube. To this was added a portion of asolution containing one of the progesterone derivatives labelled withhorseradish peroxidase, and the labelled antigen was allowed to reactwith the insoluble antibody for 60 minutes. A portion of a standardprogesterone solution (0.2, 1, 10, 30 or 100 ng/ml) was then added tothe reaction system and the immune reaction was performed for 20minutes, followed by a washing process. Thereafter, the enzyme(peroxidase) reaction was performed for 10 minutes in the presence ofhydrogen peroxide as the substrate and orthophenylenediamine as thecolor reagent, and then the absorbance of the colored substance wasmeasured.

To another glass tube in which the purified monoclonal anti-progesteroneantibody had been immobilized was added the solution containing theperoxidase-labelled progesterone derivative, followed by the immediateaddition of the standard progesterone solution by eliminating the stepfor the reaction of the insoluble antibody with the labelled antigen.After performing 20 minutes of the immune reaction, the enzyme reactionwas performed in the same manner as described above, and the result wascompared to that of the above result.

Examples of the results are shown in FIGS. 5a, 5b, 5c and 5d, whereinP7-006 (immunogen, progesterone-11α-hemisuccinate.bovine serum albumin)or P15-037 (immunogen, progesterone-11α-hemisuccinate.bovine serumalbumin) was used as the antibody andprogesterone-11α-hemisuccinate.peroxidase orprogesterone-19-hemisuccinate.peroxidase was used as the labelledprogesterone derivative.

It was found that a combination of the antibody P7-006 and theperoxidase-labelled progesterone-19-hemisuccinate was appropriate forthe measurement of blood progesterone. It was found also that P6-057(immunogen, progesterone-11α-hemisuccinate.bovine serum albumin) andP17-016 (immunogen, progesterone-11α-hemisuccinate.bovine serum albumin)were appropriate for the measurement of blood progesterone (data notshown).

FIG. 6 shows changes in the immune reaction during the reaction periodafter the addition of a standard progesterone solution, in the case of acombination of the antibody P7-006 and the peroxidase-labelledprogesterone-19-hemisuccinate.

Based on the result as shown in FIG. 6, it was confirmed that thereaction of the antibody with the peroxidase-labelled progesterone isreversible and the immune reaction, in which the progesterone to bemeasured in the sample (standard progesterone solution) and the labelledprogesterone derivative bind competitively to the antibody, reaches itsequilibrium state quickly (about 20 minutes).

Example 10

Measurement of urinary estrogen.

Antibody E15-008 was immobilized in a glass tube (diameter, 10 mm;length, 65 mm). A 200 μl portion of estrone-17-carboxymethyloxime.peroxidase solution, with its concentration of 50 ng/ml calculatedin terms of peroxidase in the compound, was transferred into the tubeand allowed to react with the insoluble antibody. After 60 minutes ofthe reaction, the resulting solution in the tube was subjected tofreeze-drying. The dried body was sealed in the tube against the accessof air, and the sealed tube was stored as a device for use in themeasurement of urinary estrogen.

Measurement of urinary estrogen was performed as follows. A portion (250μl under normal conditions) of a sample, which is diluted in advance ifnecessary, or a standard estrogen solution, was transferred into theabove-described device (test tube) for the measurement of urinaryestrogen. After 20 minutes of the immune reaction and subsequent washingprocess, the enzyme (peroxidase) reaction was performed for 10 minutesby adding into the test tube a 500 μl portion of a substrate solutionwhich consisted of orthophenylenediamine (0.3%) as a color reagent andhydrogen peroxide (0.027%) as the substrate. The enzyme reaction wasstopped by adding 1000 μl of 3M phosphoric acid solution (sulfuric acid,hydrochloric acid and the like can be used as well), and the absorbanceof the colored agent was measured at 492 nm. The concentration ofestrogen in the sample was calculated from a standard curve.

The concentration of estrogen in the same sample was separately measuredby using commercially available assay kits, and correlation coefficientsbetween the measuring method of the present invention and thesecommercial kits were calculated. The assay kits used wereRadioimmunoassay kit (from Amersham Ltd.), Fluorescence PolarizationImmunoassay (from Abbott Lab.) and E₃ Kit (a color reaction systemproduced by Teikoku Hormone Mfg. Co., Ltd. ). A total of 54 samples werechecked.

As shown in Table 6, a high correlation was found between the dataobtained by using the reagent for immunoassay and the immunoassay deviceof the present invention and the data obtained by using any of theabove-described commonly used measuring methods.

                  TABLE 6                                                         ______________________________________                                                        Correlative   Correlation                                     Prior art       relation      coefficient                                     ______________________________________                                        Radioimmunoassay                                                                              y = 0.95X - 1.09                                                                            r = 0.96                                        (Amersham)                                                                    Fluorescence Polarization                                                                     y = 0.86X - 2.39                                                                            r = 0.97                                        Immunoassay (Abbott)                                                          Color reaction (E.sub.3 Kit,                                                                  y = 1.12X - 2.74                                                                            r = 0.94                                        Teikoku Hormone Mfg.)                                                         ______________________________________                                    

Example 11

Measurement of blood estradiol.

Antibody E₂ 13-074 was immobilized in a glass tube (diameter, 10 mm;length, 65 mm). A 200 μl portion ofestradiol-6α-hemisuccinate-peroxidase solution, with its concentrationof 10 ng/ml calculated in terms of peroxidase in the compound, wastransferred into the tube and allowed to react with the insolubleantibody. After 60 minutes of the reaction, the resulting solution inthe tube was subjected to freeze-drying. The dried body was sealed inthe tube the access of air, and the sealed tube was stored as a devicefor use in the measurement of blood estradiol.

Measurement of blood estradiol was performed as follows. A portion (250μl under normal conditions) of a sample, which is diluted in advance ifnecessary, or a standard estradiol solution, was transferred into theabove-described device (test tube) for the measurement of bloodestradiol. After 20 minutes of the immune reaction and subsequentwashing process, the enzyme (peroxidase) reaction was performed for 10minutes by adding into the test tube a 500 μl portion of a substratesolution which consisted of orthophenylenediamine (0.3%) as a colorreagent and hydrogen peroxide (0.027%) as the substrate. The enzymereaction was stopped by adding 1000 μl of 3M phosphoric acid solution(sulfuric acid, hydrochloric acid and the like can be used as well), andthe absorbance of the colored solution was measured at 492 nm. Theconcentration of estradiol in the sample was calculated from a standardcurve.

The concentration of estradiol in the same sample was separatelymeasured by using commercially available assay kits, and correlationcoefficients between the measuring method of the present invention andthese commercial kits were calculated. The assay kits used wereRadioimmunoassay (produced by Diagnostic Products Corporation) andRadioimmunoassay (I-125 Kit, produced by Commissariant A LenegieAtomique). A total of 34 samples were checked.

As shown in Table 7, a high correlation was found between the dataobtained by using the reagent for immunoassay and the immunoassay deviceof the present invention and the data obtained by using any of theabove-described commonly used measuring methods.

                  TABLE 7                                                         ______________________________________                                                       Correlative   Correlation                                      Prior art      relation      coefficient                                      ______________________________________                                        Radioimmunoassay                                                                             y = 1.13X + 2.12                                                                            r = 0.93                                         (DPC)                                                                         Radioimmunoassay                                                                             y = 1.08X - 3.62                                                                            r = 0.98                                         (I-125 Kit, CIS)                                                              ______________________________________                                    

Example 12

Measurement of blood progesterone.

Antibody P7-006 was immobilized in a glass tube (diameter, 10 mm;length, 65 mm). A 200 μl portion ofprogesterone-19-hemisuccinate.peroxidase solution, with itsconcentration of 20 ng/ml calculated in terms of peroxidase in thecompound, was transferred into the tube and allowed to react with theinsoluble antibody. After 60 minutes of the reaction, resulting solutionin the tube was subjected to freeze-drying. The dried body was sealed inthe tube against the access of air, and the sealed tube was stored as adevice for use in the measurement of blood progesterone.

The measurement of blood progesterone was performed as follows. Aportion (250 μl in normal conditions) of a sample, which is diluted inadvance if necessary, or a standard progesterone, was transferred intothe above-described device (test tube) for the measurement of bloodprogesterone. After 20 minutes of the immune reaction and subsequentwashing process, the enzyme (peroxidase) reaction was performed for 10minutes by adding into the test tube a 500 μl portion of a substratesolution which consisted of orthophenylenediamine (0.3%) as a colorreagent and hydrogen peroxide (0.027%) as the substrate. The enzymereaction was stopped by adding 1000 μl of 3M phosphoric acid solution(sulfuric acid, hydrochloric acid and the like can be used as well), andthe absorbance of the colored solution was measured at 492 nm. Theconcentration of progesterone in the sample was calculated from astandard curve.

The concentration of progesterone in the same sample was separatelymeasured by using commercially available assay kits, and correlationcoefficients between the measuring method of the present invention andthese commercial kits were calculated. The assay kits used wereRadioimmunoassay (produced by Diagnostic Products Corporation) andRadioimmunoassay (produced by Daiichi Radioisotope Labs., LTD.). A totalof 54 samples were checked.

As shown in Table 8, a high correlation was found between the dataobtained by using the reagent for immunoassay use and the immunoassaydevice of the present invention and the data obtained by using any ofthe above-described commonly used measuring methods.

                  TABLE 8                                                         ______________________________________                                                        Correlative   Correlation                                     Prior art       relation      coefficient                                     ______________________________________                                        Radioimmunoassay                                                                              y = 1.01X - 0.76                                                                            r = 0.93                                        (DPC)                                                                         Radioimmunoassay                                                                              y = 1.09X - 0.12                                                                            r = 0.98                                        (Daiichi Radioisotope                                                         Labs., LTD.)                                                                  ______________________________________                                    

Thus, it is apparent that there has been provided, in accordance withthe present invention, a reagent for use in an immunoassay and animmunoassay device in which the reagent is included in a container, formeasuring haptens, antigens or antibodies by means of a competitivebinding method.

The immunoassay device of the present invention renders possible easyand simple manual operation and simplification of the mechanicalstructure of an automatic measuring apparatus, because a process for thesupply of a labelled substance is not necessary.

In addition, though the measuring principle according to the presentinvention is based on the competitive binding method, the reaction aresimilar to those steps for the sandwich method-based immunoassay.Consequently, by the use of the immunoassay device of the presentinvention, immunoassay techniques may be carried out more easily, andboth the competitive binding method and the sandwich method could beapplied to a single automatic measuring apparatus.

While the present invention has been described in conjunction withspecific embodiments and examples thereof, it is evident that manyalternatives, modifications and variations will be apparent to thoseskilled in the art in light of the foregoing description. Accordingly,it is intended to include all such alternatives, modifications andvariations as set forth within the spirit and scope of the appendedclaims.

What is claimed is:
 1. A reagent for use in an immunoassay for measuringestrogen or estradiol, comprising:(a) a labelled estrogen or estradiolderivative comprising a first estrogen or estradiol derivative and alabelling agent bound to said first estrogen or estradiol derivative,and (b) an antibody prepared by using an immunogen comprising a secondestrogen or estradiol derivative and an immunoactive carrier bound tosaid second estrogen or estradiol derivative, wherein said firstestrogen or estradiol derivative of (a) and said second estrogen orestradiol derivative of (b) are analogous substances that differ fromeach other in having different chemical structures, respectively, sothat said labelled estrogen or estradiol derivative of (a) and saidantibody of (b) can undergo reversible binding-unbinding, said labelledestrogen or estradiol derivative of (a) and said antibody of (b) beingpreliminarily bound before contacting said estrogen or estradiol to bemeasured and then unbound after contacting said estrogen or estradiol tobe measured, facilitating measurement of said estrogen or estradiol tobe measured in said immunoassay, and wherein said first and secondestrogen or estradiol derivatives are individually selected from thegroup consisting of compounds represented by formulae I to IV: ##STR8##wherein R' is hydrogen and R² is Y¹ --Z; or R¹ is Y² --Z and R² isoxygen; ##STR9## wherein R³, R⁴ and R⁵ are hydrogen, one of R⁶ and R⁷ ishydrogen, and the other is Y³ --Z; R³ is Y² --Z, R⁴, R⁵ and R⁷ arehydrogen, and R⁶ is Y³ --Z; R³ and R⁷ are hydrogen, one of R⁴ and R⁵ ishydrogen and the other is Y³ --Z, and R⁶ is hydroxyl; or R³ and R⁷ arehydrogen, R⁴ and R⁵ together represent Y¹ --Z bonded to the steroidnucleus by a double bond, and R⁶ is hydroxyl; ##STR10## wherein R⁸ ishydrogen, R⁹ is Y³ --Z and one of R¹⁰ and R¹¹ is Y³ --Z, and the otheris hydrogen; R⁸ is hydrogen, one of R⁹ and R¹⁰ is hydroxyl and the otheris Y³ --Z, and R¹¹ is hydrogen; or R⁸ is Y² --Z, R⁹ is Y³ --Z, R¹⁰ is Y⁴--Z, and R¹¹ is hydrogen; and ##STR11## wherein one of R¹², R¹³, R¹⁴,R¹⁵, R¹⁶ and R¹⁷ is Y³ --Z and the others are hydrogen; or R¹² and R¹³,R¹⁴ and R¹⁵, or R¹⁶ and R¹⁷ together represent Y¹ --Z bonded to thesteroid nucleus by a double bond, and the others are hydrogen; whereinY¹ is a straight-chain, branched, or cyclic radical intervening Z andthe steroid nucleus comprising a backbone having 1 to 10 carbon atomsand/or hetero atoms, bonded to the steroid nucleus by a double bond; Y²is a straight-chain, branched, or cyclic radical intervening Z and theoxygen comprising a backbone having 0 to 10 carbon atoms and/or heteroatoms bonded to the oxygen by a single bond; Y³ and Y⁴ are individuallya straight-chain, branched, or cyclic radical intervening Z and thesteroid nucleus comprising a backbone having 0 to 10 carbon atoms and/orhetero atoms, bonded to the steroid nucleus by a single bond; and Z is aradical selected from the group consisting of hydrogen, --NH₂, --SH,--COOH, --CHO and ##STR12##
 2. A reagent for use in an immunoassay formeasuring an estrogen or estradiol according to claim 1, wherein R¹, R³and R⁸ are hydrogen or CH₂ --COOH; R² is oxygen or N--O--CH₂ --COOH; R⁴and R⁵ are hydrogen, O--CO--CH₂ --CH₂ --CH₂ --COOH, O--CO--CH₂ --CH₂--COOH, O--CH₂ --COOH, O--CH₂ --CO--NH--CH₂ --CH₂ --NH₂ or ##STR13## orR⁴ and R⁵ taken together represent N--O--CH₂ --COOH bonded to thesteroid nucleus by a double bond; and R⁶, R⁷, R⁹, R¹⁰ and R¹¹ arehydrogen, OH, O--CO--CH₂ --CH₂ --COOH, O--CO--CH₂ --CH₂ --CH₂ --COOH or##STR14## R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷ are hydrogen, O--CO--CH₂ --CH₂--COOH, O--CO--CH₂ --CH₂ --CH₂ --COOH or S--CH₂ --COOH.
 3. A reagent foruse in an immunoassay for measuring an estrogen or estradiol accordingto claim 1, wherein said first and second estrogen or estradiolderivatives are individually selected from the group consisting ofestrone, estrone-17-carboxymethyl oxime, estrone-3-carboxymethyl ether,estradiol, estradiol-3-carboxymethyl ether, estradiol-6α-hemisuccinate,estradiol-6-carboxymethyl oxime, estradiol-17α-hemisuccinate andestradiol-17β-hemisuccinate, estriol, estriol-3-carboxymethyl ether,estriol-16α,17β-dihemisuccinate, estriol-16β,17β-dihemisuccinate,estriol-16α-glucuronide, estriol-17β-glucuronide,estradiol-7-hemisuccinate, estradiol-7-carboxyethyl thioether,estradiol-11-hemisuccinate and estradiol-16α-hemisuccinate.
 4. A reagentfor use in an immunoassay for measuring estrogen or estradiol,comprising:(c) a first estrogen or estradiol derivative, and (d) alabelled antibody comprising an antibody (b) prepared by using animmunogen comprising a second estrogen or estradiol derivative and animmunoactive carrier bound to said second estrogen or estradiolderivative, and a labelling agent bound to said antibody, wherein saidfirst estrogen or estradiol derivative of (c) and said labelled antibodyof (d) can undergo reversible binding, and said first estrogen orestradiol derivative of (c) and said second estrogen or estradiolderivative of (d) of the immunogen used for preparing said antibody (b)of the labelled antibody of (d) are analogous substances that differfrom each other in having different chemical structures, respectively,so that said first estrogen or estradiol derivative of (c) and saidlabelled antibody of (d) can undergo reversible binding-unbinding, saidfirst estrogen or estradiol derivative of (c) and said labelled antibodyof (d) being preliminarily bound before contacting said estrogen orestradiol to be measured and then unbound after contacting saidestradiol to be measured, facilitating measurement of said estrogen orestradiol to be measured in said immunoassay, wherein said first andsecond estrogen or estradiol derivatives are individually selected fromthe group consisting of compounds represented by formulae I to IV:##STR15## wherein R¹ is hydrogen and R² is Y¹ --Z; or R¹ is Y² --Z andR² is oxygen; ##STR16## wherein R³, R⁴ and R⁵ are hydrogen, one of R⁶and R⁷ is hydrogen, and the other is Y³ --Z; R³ is Y² --Z, R⁴, R⁵ and R⁷are hydrogen, and R⁶ is Y³ --Z; R³ and R⁷ are hydrogen, one of R⁴ and R⁵is hydrogen and the other is Y³ --Z, and R⁶ is hydroxyl; or R³ and R⁷are hydrogen, R⁴ and R⁵ together represent Y¹ --Z bonded to the steroidnucleus by a double bond, and R⁶ is hydroxyl; ##STR17## wherein R⁸ ishydrogen, R⁹ is Y³ --Z and one of R¹⁰ and R¹¹ is Y³ --Z, and the otheris hydrogen; R⁸ is hydrogen, one of R⁹ and R¹⁰ is hydroxyl and the otheris Y³ --Z, and R¹¹ is hydrogen; or R⁸ is Y² --Z, R⁹ is Y³ --Z, R¹⁰ is Y⁴--Z, and R¹¹ is hydrogen; and ##STR18## wherein one of R¹², R¹³, R¹⁴,R¹⁵, R¹⁶ and R¹⁷ is Y³ --Z and the others are hydrogen; or R¹² and R¹³,R¹⁴ and R¹⁵, or R¹⁶ and R¹⁷ together represent Y¹ --Z bonded to thesteroid nucleus by a double bond, and the others are hydrogen; whereinY¹ is a straight-chain, branched, or cyclic radical intervening Z andthe steroid nucleus comprising a backbone having 1 to 10 carbon atomsand/or hetero atoms, bonded to the steroid nucleus by a double bond; Y²is a straight-chain, branched, or cyclic radical intervening Z and theoxygen comprising a backbone having 0 to 10 carbon atoms and/or heteroatoms bonded to the oxygen by a single bond; Y³ and Y⁴ are individuallya straight-chain, branched, or cyclic radical intervening Z and thesteroid nucleus comprising a backbone having 0 to 10 carbon atoms and/orhetero atoms, bonded to the steroid nucleus by a single bond; and Z is aradical selected from the group consisting of hydrogen, --NH₂, --SH,--COOH, --CHO and ##STR19##
 5. A reagent for use in an immunoassay formeasuring an estrogen or estradiol according to claim 4, wherein R¹, R³and R⁸ are hydrogen or CH₂ --COOH; R² is oxygen or N--O--CH₂ --COOH; R⁴and R⁵ are hydrogen, O--CO--CH₂ --CH₂ --CH₂ --COOH, O--CO--CH₂ --CH₂--COOH, O--CH₂ --COOH, O--CH₂ --CO--NH--CH₂ --CH₂ --NH₂ or ##STR20## orR⁴ and R⁵ taken together represent N--O--CH₂ --COOH bonded to thesteroid nucleus by a double bond; and R⁶, R⁷, R⁹, R¹⁰ and R¹¹ arehydrogen, OH, O--CO--CH₂ --CH₂ --COOH, O--CO--CH₂ --CH₂ --CH₂ --COOH or##STR21## R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷ are hydrogen, O--CO--CH₂ --CH₂--COOH, O--CO--CH₂ --CH₂ --CH₂ --COOH or S--CH₂ --COOH.
 6. A reagent foruse in an immunoassay for measuring an estrogen or estradiol accordingto claim 4, wherein said first and second estrogen or estradiolderivatives are individually selected from the group consisting ofestrone, estrone-17-carboxymethyl oxime, estrone-3-carboxymethyl ether,estradiol, estradiol-3-carboxymethyl ether, estradiol-6α-hemisuccinate,estradiol-6-carboxymethyl oxime, estradiol-17α-hemisuccinate andestradiol-17β-hemisuccinate, estriol, estriol-3-carboxymethyl ether,estriol-16α, 17β-dihemisuccinate, estriol-16β, 17β-dihemisuccinate,estriol-16α-glucuronide, estriol-17β-glucuronide,estradiol-7-hemisuccinate, estradiol-7-carboxyethyl thioether,estradiol-11-hemisuccinate and estradiol-16α-hemisuccinate.
 7. Thereagent for use in an immunoassay for measuring estrogen or estradiolaccording to claim 1,wherein said first estrogen or estradiol derivativeof (a) and said second estrogen or estradiol derivative of (b) are acombination selected from the group consisting of(1) a combination ofestrone-17-carboxymethyloxime and estriol-16α, 17β-dihemisuccinate, (2)a combination of estrone-17-carboxymethyloxime andestradiol-17β-hemisuccinate, (3) a combination ofestrone-17-carboxymethyloxime and estriol-16α-glucuronide, (4) acombination of estriol-16α-glucuronide and estriol-16α,17β-dihemisuccinate, (5) a combination of estriol-16α-glucuronide andestradiol-17β-hemisuccinate, (6) a combination ofestradiol-17α-hemisuccinate and estriol-16α, 17β-dihemisuccinate, (7) acombination of estradiol-17α-hemisuccinate andestradiol-17β-hemisuccinate, (8) a combination ofestradiol-17α-hemisuccinate and estriol-16α-glucuronide, and (9) acombination of estriol-16β, 17β-dihemisuccinate and estriol-16α,17β-dihemisuccinate.
 8. The reagent for use in an immunoassay formeasuring estrogen or estradiol according to claim 1,wherein said firstestrogen or estradiol derivative of (a) and said second estrogen orestradiol derivative of (b) are a combination selected from the groupconsisting of(1) a combination of estradiol-6α-hemisuccinate andestradiol-6-carboxymethyloxime, (2) a combination ofestradiol-6α-hemisuccinate and estradiol-3-carboxymethylether, (3) acombination of estradiol-6-carboxymethyloxime andestradiol-3-carboxymethylether, (4) a combination ofestradiol-6-carboxymethyloxime and estradiol-6α-hemisuccinate, (5) acombination of estradiol-3-carboxymethylether andestradiol-6-carboxymethyloxime, and (6) a combination ofestradiol-3-carboxymethylether and estradiol-6α-hemisuccinate.
 9. Thereagent for use in an immunoassay for measuring an estrogen or estradiolaccording to claim 1,wherein said first estrogen or estradiol derivativeof (a) and said second estrogen or estradiol derivative of (b) are acombination selected from the group consisting of(1) a combination ofestrone-17-carboxymethyloxime and estriol-16α, 17β-dihemisuccinate, (2)a combination of estrone-17-carboxymethyloxime andestradiol-17β-hemisuccinate, (3) a combination ofestriol-16α-glucuronide and estriol-16α, 17β-dihemisuccinate, and (4) acombination of estriol-16α-glucuronide and estradiol-17β-hemisuccinate.10. The reagent for use in an immunoassay for measuring estrogen orestradiol according to claim 1,wherein said first estrogen or estradiolderivative of (a) and said second estrogen estradiol derivative of (b)are a combination selected from the group consisting of(1) a combinationof estradiol-6α-hemisuccinate and estradiol-6-carboxymethyloxime, and(2) a combination of estradiol-3-carboxymethylether andestradiol-6-carboxymethyloxime.
 11. The reagent for use in animmunoassay for measuring estrogen or estradiol according to claim4,wherein said first estrogen or estradiol derivative of (c) and saidsecond estrogen or estradiol derivative of (d) are a combinationselected from the group consisting of(1) a combination ofestrone-17-carboxymethyloxime and estriol-16α, 17β-dehemisuccinate, (2)a combination of estrone-17-carboxymethyloxime andestradiol-17β-hemisuccinate, (3) a combination ofestrone-17-carboxymethyloxime and estriol-16α-glucuronide, (4) acombination of estriol-16α-glucuronide and estriol-16α,17β-dihemisuccinate, (5) a combination of estriol-16α-glucuronide andestradiol-17β-hemisuccinate, (6) a combination ofestradiol-17α-hemisuccinate and estriol-16α, 17β-dihemisuccinate, (7) acombination of estradiol-17α-hemisuccinate andestradiol-17β-hemisuccinate, (8) a combination ofestradiol-17α-hemisuccinate and estriol-16α-glucuronide, and (9) acombination of estriol-16β, 17β-dihemisuccinate and estriol-16α,17β-dihemisuccinate.
 12. The reagent for use an immunoassay formeasuring estrogen or estradiol according to claim 4,wherein said firstestrogen or estradiol derivative of (c) and said second estrogen orestradiol derivative of (d) are a combination selected from the groupconsisting of(1) a combination of estradiol-6α-hemisuccinate andestradiol-6-carboxymethyloxime, (2) a combination ofestradiol-6α-hemisuccinate and estradiol-3-carboxymethylether, (3) acombination of estradiol-6-carboxymethyloxime andestradiol-3-carboxymethylether, (4) a combination ofestradiol-6-carboxymethyloxime and estradiol-6α-hemisuccinate, (5) acombination of estradiol-3-carboxymethylether andestradiol-6-carboxymethyloxime, and (6) a combination ofestradiol-3-carboxymethylether and estradiol-6α-hemisuccinate.
 13. Thereagent for use in an immunoassay for measuring estrogen or estradiolaccording to claim 4,wherein said first estrogen or estradiol derivativeof (c) and said second estrogen or estradiol derivative of (d) are acombination selected from the group consisting of(1) a combination ofestrone-17-carboxymethyloxime and estriol-16α, 17β-dihemisuccinate, (2)a combination of estrone-17-carboxymethyloxime andestradiol-17β-hemisuccinate, (3) a combination ofestriol-16α-glucuronide and estriol-16α, 17β-dihemisuccinate, and (4) acombination of estriol-16α-glucuronide and estradiol-17β-hemisuccinate.14. The reagent for use in an immunoassay for measuring estrogen orestradiol according to claim 4,wherein said first estrogen or estradiolderivative of (c) and said second estrogen or estradiol derivative of(d) are a combination selected from the group consisting of(1) acombination of estradiol-6α-hemisuccinate andestradiol-6-carboxymethyloxime, and (2) a combination ofestradiol-3-carboxymethylether and estradiol-6-carboxymethyloxime.